Peptidases (proteolytic enzymes) constitute 1%-5% of eukaryotic genes, and are essential for many biological processes. Peptidases of the S9B/DPPIV family are unique in their ability to cleave off N-terminal dipeptides from substrates that have a proline residue at the second position (XaaPro). Four active members of this family are known, two of which are cell surface peptidases: Dipeptidyl peptidase IV (DPPIV) and the Fibroblast activation protein alpha (FAP).
The two cytosolic members of the DPPIV family are DPP8 and DPP9, which are expressed in multiple tissues, predominantly in lymphocytes and epithelial cells of many organs (Yu et al., 2009; Tang et al., 2009; Schade et al., 2008). DPP8 and DPP9 share overall 27% and 38% amino-acid identity (respectively) with DPPIV. Moreover, DPP8 and DPP9 are highly homologues (57% identical) sharing over 90% amino acid identity within their active sites (Van Goethem et al., 2011; Abbott et al., 2000; Ajami et al., 2004). In vitro, DPP8 and DPP9 are similar in their biochemical properties, including enzyme kinetics and substrate specificity (Geiss-Friedlander et al., 2009).
The physiological roles of DPP8 and DPP9 are only emerging. Down-regulation of both peptidases in several cell lines using siRNA treatment, shows that DPP9 but not DPP8, is rate-limiting for degradation of most cytosolic proline containing peptides (Geiss-Friedlander et al., 2009). Changes in the expression levels of DPP8 and DPP9 are critical for survival and proliferation of different cell lines, such as human hepatoma and embryonic kidney cells as well as cells originating from the Ewing sarcoma family of tumors (Yao et al., 2011; Lu et al., 2011). Over-expression of both peptidases was reported to impair cell adhesion and migration (Yu et al., 2006).
DPP8 and DPP9 have been shown to influence cell behavior such as cell-extracellular matrix interactions, proliferation and apoptosis. DPP9 transcription is abundant in human tumor cell lines such as melanoma, chronic myelogenous leukemia, colorectal adenocarcinoma, neuroblastoma, and HeLa cells (a cell line derived from cervical cancer), and is upregulated in human testicular tumors, as well as breast cancer cell lines, in particular estrogen negative breast cancer cell lines. DPP8 transcription as compared to transcription of other DPPs is significantly increased in breast cancer and ovarian cancer cell lines. Constitutive expression of DPP8 and DPP9 was also found in B cell chronic lymphocytic leukemia. The expression of DPP8 and DPP9 in tumor tissues and cell lines indicates that they may have roles in tumor pathogenesis (reviewed in Zhang et al., 2013). Inhibition of DPP8 and DPP9 by enzyme inhibition or siRNA breakdown have been shown to enhance cell death and tumor regression.
All inhibitors described so far for the DPP family, are competitive inhibitors that target the active site of the peptidase, which is highly conserved, making it difficult to target one specific member of the family.
Inhibitors for DPP IV are described in WO 2011/113895, US 2011/0218142 and US 2008/0293618. WO 2005/106487 describes antibodies and siRNA for modulating DPP9 activity.
SUMOs are small proteins that act as post-translational protein modifiers. Modification of proteins by SUMO (sumoylation) affects many cellular pathways including cell cycle progression, chromatin structure, DNA repair, transcription and trafficking. Humans express three functional SUMO homologs: SUMO1-3, which are conjugated to their targets in a reversible manner. SUMO2 and SUMO3 are highly homologous (97% identity) but share only 50% identity with SUMO1. The three homologs appear to serve overlapping but also distinct functions, since some proteins are modified preferably by one of the SUMO homolog (Geiss-Friedlander & Melchior, 2007).
Sumoylation can lead to various outcomes including changes in the localization, activity, solubility, or even stability of respective target proteins. These are due to changes in the molecular interactions of the modified proteins. Consequently, novel interactions depend on the presence of downstream effector proteins that contain motifs for non-covalent binding to SUMO. A single SUMO-interacting motif, SIM, is currently known which is characterized by a cluster of hydrophobic residues (Song et al., 2004; Hannich et al., 2005; Hecker et al., 2006). NMR and crystal structures reveal that SIMs bind to a SIM-interacting groove (SIG) formed between the α-helix and β-sheet of all three SUMO homologs (Song et al., 2004; Hannich et al., 2005; Hecker et al., 2006; Baba et al., 2005; Reverter & Lima, 2005). How SUMO-interacting proteins differentiate between the SUMO homologs is only partially understood. In some cases negative charges flanking the SIM, lead to a preferable interaction of the SIMs with SUMO1 over SUMO2/3 (Hecker et al., 2006; Chang et al., 2011).